Cells in the body are continually replaced. This can occur over a period of several hours up to many months. The presence of stem cells of various tissue types are present to grow and differentiate as a source of replacement cells for those cells that die. For example, the majority of blood cells are destined to die within a period of hours to weeks, depending on the specific blood cell type, and so must be continuously replaced. Bone Marrow (BM) is the major source of blood cells, including both white (lymphocytes, i.e., immune cells) and red blood cells (erythrocytes) in the adult.
Among the various cells that constitute the BM are primitive hematopoietic pluripotent stem cells, progenitor cells and stromal cells. Hemopoietic stem cells are a class of cells that have been defined functionally by the characteristics of extended self-renewal and the capacity to mature into one or more differentiated forms of blood cells, such as, lymphocytes, granulocytes, erythrocytes, megakaryocytes and stromal cells. Hence, an important property of stem cells is their ability to both proliferate, which ensures a continuous supply throughout the lifetime of an individual, and differentiate into the mature cells of the peripheral blood system.
Hematopoietic stem cells are present at about 0.01% of the bone marrow and serve as a source of replacement blood cells. When necessary, a pluripotent stem cell can begin to differentiate, and after successive divisions become committed, thus losing the capacity for self-renewal, to a particular line of development. A pluripotent hemopoietic stem cell, such as long term culture initiating cells (LTC-IC), can differentiate into either a lymphoid stem cell or a myeloid stem cell. The LTC-IC cells are one of the most primitive of cells and can potentially differentiate in any hematopoietic lineage.
Lymphoid stem cells can differentiate into a B-cell progenitor or a T-cell in the thymus. The B-cell progenitor formation is induced by Interleukin-7 (IL-7) or other differentiation factors, and is inhibited by TGF-β or Interleukin-4 (IL-4). The lymphoid stem cell can also differentiate into a T-cell progenitor cell that moves to the thymus wherein it differentiates into a pre-T-cell, followed by differentiation into a specific T-cell.
Myeloid stem cells may differentiate into a burst-forming unit-erythroid followed by a colony forming unit-erythroid or by further differentiation into a red blood cell induced by erythropoietin. Alternatively, a myeloid stem cell can differentiate into a colony forming unit-megakaryocyte followed by differentiation into a megakaryocyte that can then form platelets. Myeloid stem cells can also differentiate into a colony forming unit granulocyte macrophage (CFU-GM), which are the progenitors of monocytes and macrophages, colony forming unit eosinophil (CFU-E) or a colony forming unit basophil (CFU-B), which can then differentiate into eosinophils or basophils, respectively, under the control of GM-CSF/IL-3 or IL-3, respectively, and CFU-C cells. On the other hand, both TNF-α, TGF-β and TGF-β1 efficiently inhibit myeloid cell development (Zipori et al., 1986 and Broxmeyer et al., 1986) and TGF-β1 restricts lymphocyte proliferation (lee et al., 1986) and is a potent inhibitor of several proliferative activators for myelocytes and lymphoblasts including IL-3, G-CSF, GM-CSF (Masatsugu et al., 1987).
Virtually all of the circulating blood cells, including erythrocytes, leukocytes or lymphocytes, granulocytes and platelets originate from various progenitor cells that are themselves derived from precursor stem cells such as lymphoid and myeloid cells. Stem cells may also differentiate in to the supporting cells of the hemopoietic bone marrow matrix, such as stromal cells, that function in the formation and makeup of the hematopoietic microenvironment, which supports growth and differentiation of other hematopoietic stem cells into the various types of lymphoid and myeloid cells under the control of cytokines (described below).
Besides providing support these cells can also secrete multiple cytokines that have effects on the growth and/or differentiation of the lymphoid and myeloid progenitor cells. In addition, other cytokines (such as colony stimulating factors) can bind to stem cell growth supporting stromal cells and influence the types and concentrations of growth and/or differentiation factors secreted by the stromal cells.
Hematopoietic stem cell production, proliferation and differentiation occur in direct proximity to osteoblasts, a major component of the bone matrix, within the bone marrow cavity. The development of the bone marrow cavity is a coordinated process in which blood precursors migrate and colonize spaces carved out of embryonic bone and cartilage. Stromal cells like osteoblasts in the bone marrow, through adhesion events mediated by cell adhesion molecules (CAMs), such as integrins and selecting, provide the structural scaffolding for hematopoiesis, and also produce several of the soluble factors (cytokines) critical to the development of blood cells. Hence, the direct interaction of hematopoietic cells with osteoblasts stromal cells is important for maintaining hematopoiesis. Stromal cell-associated cytokines are expressed due to this interaction and stimulate blood cell proliferation and are involved in controlling the specific growth and differentiation of hematopoictic stem cells into the specific blood cell types.
The morphologically recognizable and functionally capable cells circulating in the blood include erythrocytes (red blood cells), leukocytes (white blood cells including both B and T cells), non B- and T-lymphocytes, phagocytes, neutrophilic, eosinophilic and basophilic granulocytes, and platelets. These mature cells are derived, on demand, from dividing progenitor cells, such as erythroblasts (for erythrocytes), lymphoid precursors, myeloblasts (for phagocytes including monocytes, macrophages and neutrophils), promyelocytes and myelocytes (for the various granulocytes) and megakaryocytes for the platelets. As stated above, these progenitor cells are themselves derived from lymphoid and myeloid precursor stem cells.
Many different proteins regulate cell viability, growth and differentiation of different hematopoietic cell lineages and play various roles in the molecular basis of abnormal cell development in blood-forming tissues. These regulators include cytokines such as colony stimulating factors and interleukins. Different cytokines can induce cell viability, multiplication and differentiation. Hematopoiesis is controlled by a network of interactions between these cytokines. This network includes positive regulators such as colony stimulating factors and interleukins and negative regulators such as transforming growth factor β (TGF-β) and tumor necrosis factors α and β (TNF). The functioning of this network requires an appropriate balance between these positive and negative regulators.
Hence, a complex network of cytokines as well as inter- and intra-cellular interactions regulate the proliferation and differentiation of a finite pool of hematopoietic stem cells. As described above, cytokines are protein growth factors that function as intercellular signals that regulate hematopoiesis, as well as local and/or systemic inflammatory responses, and include colony stimulating factors, interleukins and other growth factors having such activity.
Cytokines modulate target cells by interacting with cytokine receptors on the target cell. Principal cell sources of cytokines in vivo include T lymphocytes, B lymphocytes, macrophages, monocytes, platelets and stromal cells. While cytokine specific receptors are specific for a given cytokine, cytokine receptors are grouped into families based on shared features. The first group of cytokine receptors is the hemopoetin group which are present on cells including immune system cells that bind IL-2, IL-3, IL-4, IL-6 and IL-7. A second receptor family is the TNF receptor family which bind both TNF-α and TGF-β. A third family is the immunoglobulin (Ig) superfamily receptor family, which contain an Ig sequence-like motif and includes human IL-1 and IL-6 receptors.
Proliferation and differentiation of hematopoietic cells are regulated by hormone-like growth and differentiation factors designated as colony-stimulating factors (CSF) (Metcalf, D. Nature 339, 27–30 (1989)). CSF can be classified into several factors according to the stage of the hematopoietic cells to be stimulated and the surrounding conditions as follows: granulocyte colony-stimulation factor (G-CSF), granulocyte-macrophage colony-stimulation factor (GM-CSF), macrophage colony-stimulation factor (M-CSF), and interleukin 3 (IL-3).
Small amounts of certain hematopoietic growth factors account for the differentiation of stem cells into a variety of blood cell progenitors, for the tremendous proliferation of those cells, and for their differentiation into mature blood cells. For instance, G-CSF participates greatly in the differentiation and growth of neutrophilic granulocytes and plays an important role in the regulation of blood levels of neutrophils and the activation of mature neutrophils (Nagata, S., “Handbook of Experimental Pharmacology”, volume “Peptide Growth Factors and Their Receptors”, eds. Sporn, M. B. and Roberts, A. B., Spring-Verlag, Heidelberg, Vol.95/1, pp.699–722 (1990); Nicola, N. A. et al., Annu. Rev. Biochem. 58, pp.45–77 (1989)). It is also reported that G-CSF stimulates the growth of tumor cells such as myeloid leukemia cells. (Nicola and Metcalf, Proc. Natl. Acad. Sci. USA, 81, 3765–3769 (1984); Begley et al., Leukemia, 1, 1–8 (1987). Other growth factors include, erythropoietin (EPO), which is responsible for stimulating the differentiation of erythroblasts into erythrocytes and Macrophage-Colony Stimulating Factor (M-CSF) responsible for stimulating the differentiation of myeloblasts and myelocytes into monocytes.
Growth factors are part of the cytokine family of chemical messengers. As stated above, cytokines are among the factors that act upon the hematopoietic system to regulate blood cell proliferation and differentiation and they are also important mediators of the immune response being secreted by both B and T cells, as well as other various lymphocytes. Cytokines encourage cell growth, promote cell activation, direct cellular traffic, act as messengers between cells of the hematopoietic system, and destroy target cells (i.e., cancer cells). Among the various components involved in the modulation and regulation of the hematopoietic system that cytokines play a role in modulating are the tachykinins.
The tachykinins are immune and hematopoietic modulators that belong to a family of peptides encoded by the preprotachykinin-I (PPT-1) gene (Quinn, J. P., C. E. Fiskerstrand, L. Gerrard, A. MacKenzie, and C. M. Payne. 2000. Molecular models to analyze preprotachykinin-A expression and function. Neuropeptides 34:292–302). The tachykinins can be released in the BM and other lymphoid organs as neurotransmitters or from the resident BM immune cells (2–6). In the BM, PPT-I and other hematopoietic growth factors regulate expression of each other through autocrine and paracrine activities. It is believed that various cytokines induce the expression of the PPT-I gene in BM mesenchymal cells (Rameshwar, P. 1997. Substance P: A regulatory neuropeptide for hematopoiesis and immune functions. Clin. Immunol. Immunopath. 85:129–133.). The tachykinin family of peptides exerts pleiotropic functions such as neurotransmission and immune/hematopoietic modulation.
PPT-1 peptides exert both stimulatory and inhibitory hematopoietic effects by interacting with different affinities to the G-protein coupled receptors: NK-1, NK-2 and NK-3 (Krause, J. E., Y. Takeda, and A. D. Hershey. 1992. Structure, functions, and mechanisms of substance P receptor action. J. Invest. Dermatol. 98:2S-7S). NK-1 and NK-2 expression has been reported in BM cells (Rameshwar, P., A. Poddar, and P. Gascon. 1997. Hematopoietic regulation mediated by interactions among the neurokinins and cytokines. Leuk. Lymphoma 28:1–10). NK-1 is induced in BM cells by cytokines and other stimulatory hematopoietic regulators. NK-2 is constitutively expressed in BM cells that are unstimulated or stimulated with suppressive hematopoietic regulators. NK-1 and NK-2 are not co-expressed in BM cells because NK-1 induction by cytokines is correlated with the down regulation of NK-2. In BM cells, NK-1 expression requires cell stimulation whereas its expression in neural tissue is constitutive (Rameshwar, P. 1997. Substance P: A regulatory neuropeptide for hematopoiesis and immune functions. Clin. Immunol. Immunopath. 85:129–133, Yao, R., P. Rameshwar, R. J. Donnelly, and A. Siegel. 1999. Neurokinin-1 expression and colocalization with glutamate and GABA in the hypothalamus of the cat. Mol. Brain Res. 71:149–158, and Abrahams, L. G., M. A. Rerutter, K. E. McCarson, and V. S. Seybold. 1999. Cyclic AMP regulates the expression of neurokinin 1 receptors by neonatal rat spinal neurons. J. Neurochem. 73:50–58.). It is believed that a particular cytokine discriminates between the expression of NK-1 and NK-2, which directs the type of BM functions: stimulatory vs. inhibitory (Rameshwar, P., A. Poddar, and P. Gascon. 1997. Hematopoietic regulation mediated by interactions among the neurokinins and cytokines. Leuk. Lymphoma 28:1–10).
Substance P (SP), the major tachykinin released in the BM, stimulates hematopoiesis through interactions with the neurokinin-1 (NK-1) receptor, which is resident on BM stroma, immune cells and other lymphoid organ cells. Hence, the expression of NK-1 determines the hematopoietic response of the tachykinins. NK-2 inhibits hematopoiesis by interacting with neurokinin-A, another tachykinin encoded for by the PPT-I gene. Recently, the present inventors have discovered that the stimulatory effects mediated by NK-1 can be changed to hematopoietic inhibition in the presence of the amino terminal of SP, a fragment found endogenously in the BM due to enzymatic digestion of SP by endogenous endopeptidases. Further, dysregulated expression of the PPT-1 gene has been associated with different pathologies such as cancer (Bost et al., 1992b; Henning et al., 1995; Ho et al., 1996; Michaels, 1998; Rameshwar et al., 1997a).
Under normal circumstances, the BM is able to respond quickly to an increased demand for a particular type of cell. The pluripotential stem cell is capable of creating and reconstituting all the cells that circulate in the blood, including both red and white blood cells and platelets. As stated, progenitor cells that derive from stem cells can replicate and differentiate rapidly. On average, 3–10 billion lymphocyte cells can be generated in an hour. The BM can increase this by 10 fold in response to need. However, in the throes of a diseased state, the BM may not produce enough stem cells, may produce too many stem cells or various ones produced may begin to proliferate uncontrollably.
Lymphoproliferative syndromes consist of several types of diseases known as leukemia and malignant lymphoma, which can further be classified as acute and chronic myeloid or lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. These diseases are characterized by the uncontrollable multiplication or proliferation of leukocytes (primarily the B-cells), myelocytes and tissue of the lymphatic system, especially lymphocyte cells produced in the BM and lymph nodes.
Traditional methods of treating these leukemia related malignancies includes chemotherapy and radiotherapy. The theory behind chemo- and radio-therapies is that they take advantage of a cancerous cell's unnatural and vastly increased cell growth cycle. Once adulthood is reached, the majority of cells within the body grow and proliferate for the purposes of replacing old and dying cells. As a consequence of this, the majority of the body's terminally differentiated cells are either not in a growth phase in their cell cycle or if they are it is at a slow rate. Hence, for example, cells of the skin and liver have a very slow growth rate, while cells of the kidneys grow so slow in the adult as to be non-existant. Cancerous cells, on the other hand, have a very rapid growth rate in which large amounts of DNA is synthesized and the cell's membrane becomes more permeable to extracellular elements. Due to this rapid growth and increased cell-surface permeability, cancerous cells are more susceptible to the effects of DNA degrading chemo- and radio-therapeutic agents that disrupt DNA synthesis and cause cell death. Because most cells of the body grow, if at all, at a slower rate they are not as susceptible to the toxic effects of chemo- and radio-therapy.
Problems arise however with regard to those cells of the body that naturally have increased rates of proliferation such as stem cells, progenitor cells and the many different hemopoietic cells of the immune system. Chemo- and radio-therapeutic agents do not distinguish between the aberrant cancerous cells and the rapid growing, but normal stem and immune cells. Hence, both tumor and healthy stem and immune cells are killed off by the administration of chemo- and radio-therapeutic toxic agents. This leads to a drop in blood count, suppression of the immune response, and to an increased risk of bacterial and/or fungal infection, which can lead to death.
Because stem and progenitor cells, like cancer cells, have an increased sensitivity to the toxic effects of chemo- and radio-therapy, and because chemo- and radio-therapeutic agents are indiscriminate when they assault cells of the body, leading to a compromised immune system, there has been, and continues to be, a long felt need for a way to protect the healthy stem and progenitor cells of the body from the toxic effects of chemo- and radio-therapy.
The present invention includes novel compositions and methods for slowing, and or turning off, stem and progenitor cell growth in a subject, specifically, in a subject about to undergo toxic cancer treatment, thus protecting these cells from the highly toxic effects associated with chemo- and radio-therapy. The Applicants have discovered that neutral endopeptidase (NEP) utilizes Substance P (SP) to produce a tetrapeptide, SP(1-4), that inhibits proliferation of lymphoid-myeloid stem and progenitor cells. It has been determined, through its interactions with TGF-β and TNF-α, that SP(1-4) can be used as an effective treatment to shield both stem and progenitor cells from the toxic effects of chemo- and radio-therapy thereby protecting a subject's immune system from being compromised and reducing the risk of bacterial or fungal infection, allowing for a greater dosage of chemo- and/or radio-therapy to be administered and/or over a longer administration period, as well as shortening the recovery period required for new stem cell growth and terminal blood cell replenishment.